The Author(s) 2018. This article is published with open access at www.chitkara.edu.in/publications.

ABSTRACT

The prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane glycoprotein with enzymatic activity similar to the family of zinc-dependent exopeptidases. This protein is of great medical and pharmacological interest as overexpression in prostate cells is related to the progression of prostate cancer; therefore, it represents an important target for the design of radiopharmaceuticals. The presence of two Zn2+ ions in the active site is crucial to the enzymatic activity and the design of high-affinity inhibitors. The amino acid residues coordinating these ions are highly conserved in PSMA orthologs from plants to mammals, and site-mutagenesis assays of these residues show a loss of enzymatic function or reduction of the kinetic parameters. In the present work, we performed molecular dynamics simulation of PSMA with the purpose of characterizing it energetically and structurally. We elucidated the differences of PSMA with its two Zn+2 ions as cofactors and without them in the free energy profile, and in four structural parameters: root mean square deviations and root mean square fluctuations by atom and amino acid residue, radius of gyration, and solvent accessible surface area.

INTRODUCTION

Prostate cancer is a global problem since it is the second type of cancer with the highest incidence and the fifth with the highest number of deaths among men, while in Mexico it is the cancer type with higher incidence and mortality rate [1]. The Prostate-specific Membrane Antigen (PSMA) is a type II transmembrane glycoprotein of 100 kDa composed of 750 amino acids with at least three functions: hydrolytic NAALADase activity, folate hydrolase and dipeptidyl peptidase IV activity [2-4]. This protein is overexpressed in poorly differentiated and metastatic cells; consequently, it is considered an important indicator of prostate cancer and a target for the development of many inhibitors [5-6]. Recently, small molecule inhibitors (SMI) targeting PSMA have been developed; these are zinc- binding compounds linked to glutamate or a glutamate isomer. Urea-based SMI (Glu-urea-R) have demonstrated to specifically bind to PSMA and inhibit its activity in the LNCaP cell line. In such compounds, Glu-urea is the binding terminal and the R-group is the coupling terminal to other chemical groups such as a linker and a chelator associated with radionuclides [7-10]. The theranostic agents are based on the use of a radionuclide with the same PSMA-targeting ligands for therapy and diagnosis; for this purpose 177Lu, 225Ac, and 131I have been used. Particularly, 177Lu associated with PSMA617 has provided a safe and effective therapy in patients with metastatic castration-resistant prostate cancer [11-14]. PSMA-617 is a ligand conformed by a DOTA chelator (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated with Glu-urea-Lys pharmacophore by a linker composed of two aromatic rings; it was designed for labeling with 177Lu and 68Ga to achieve high-quality image and efficient endotherapy [15-16]. Crystallographic structural studies of PSMA made possible to elucidate the interaction of the protein with the inhibitors. Structural information of PSMA is available only for the extracellular part of the protein (residues 44-750). It reveals that the protein exists as a symmetrical homodimer in vivo, each polypeptide monomer having three structural domains: a protease-like domain (residues 56-116 and 352591), an apical domain –also called the protease-associated domain- (residues 117-351), and the helical domain – also called the C-terminal domain- (residues 592-750). [Figure. 1]. The active site of the protein contains a binuclear Zn active site, catalytic residues, and a substrate-binding arginine-rich patch. A water ligand bridges the two zinc atoms, each coordinated by endogenous ligands: Zn(1) by His553 and Glu425, Zn(2) by His377 and Asp453, both atoms bounded by Asp387. Glu424 and Tyr552 have the catalytic function. A substrate/inhibitor-binding cavity with an area of about 1100 A2, and a diameter and deep of about 20 Å, is formed in the interface between the three domains; this interface is considered large as it buries around 4600 A2. It is localized in the helical domain and is formed by two pockets, S1’(pharmacophore) and S1(non-pharmacophore). The cavity has an arginine patch (Arg463, Arg534, and Arg536) involved in the right orientation of the substrate for catalysis; it is aligned with the S1 which has a chlorine ion that keeps Arg534 in a conformation that allows the interaction with the substrate, while Arg536 and Arg463 are flexible conferring tolerance to different chemical groups [Figure 2]. The “glutarate sensor” is responsible of detecting the absence or presence of glutamate in S1’ pocket and is formed by residues 692-704 together with Lys699 and Tyr700, which are also important for the specific binding of glutamate along with Arg210 in the apical domain [17-19]. The pharmacophore Glu-urea-Lys (iPSMA) [Figure 3] is capable of binding with PSMA because it has three carboxylic acid groups. The glutamate-urea fraction of the inhibitor has a predisposition to be oriented towards S1’, and lysine is used for conjugation or derivatization, through the free amine, with a linker region or a chelating agent residing in S1. When both pockets are occupied, the hydrophobic contact (due to an aromatic agent) increases resulting in higher affinity [20,21]. The affinity to PSMA is due to the binding of glutamate by its α-carboxylate, which forms a bridge with the guanidinium group of Arg210, and hydrogen bonds with the hydroxide groups of Tyr552 and Tyr700, while the γ-carboxylate interacts with Lys699 and Ans257. The catalytic activity is carried out by Glu424, that extracts a proton from the water molecule situated between the zinc atoms and activates it [19,22]. The urea group serves as a zinc-binding group (ZBG) because the oxygen of the molecule interacts with Tyr552, His553, the active water molecule and Zn(1), while N groups form hydrogen bonds: N(1) with the main carbonyl chain of Gly-518 and the carboxylate of Glu-424, and N(2) with γ-carbonyl of Gly-518 [19, 23-24].

In silico analysis of the PSMA showed that its structural stability has a direct and intrinsic dependence of its active center, in fact, the tests performed on the protein excluding the Zn atoms are conclusive to affirm that PSMA not only loses affinity to bind with molecules like inhibitors, also their energy capacity is diminished; however, there are regions between residues 100-122 and 640-730 that exhibit structural stability regardless of the absence of the Zn atoms. The tests carried out on PSMA with and without the heart of its active center, the Zn atoms, allowed us to conceive the protein as a closed system, with less energy and flexibility (in the absence of atoms). This situation is completely opposite when they are incorporated, which can be interpreted that PSMA behaves like a biological trap dependent on Zn. The CHARMM force field used was appropriate for modeling the PSMA interactions and can be adapted to study the processes involved in the active center in the presence of a urea-based inhibitor and, of course, a therapeutic radiopharmaceutical.